Molecular pharming has attracted extensive attention in production of various pharmaceutical proteins, including enzymes, vaccines, antibodies, hormones, etc. Edible mushrooms are regarded as appropriate hosts for production of recombinant proteins, especially for the development of edible vaccines. The use of mushrooms for molecular pharming has all the advantages of plant-based systems coupled with unique benefits including complete duplication, fast growth, scale-up production under controlled conditions and less gene contamination. However, the success of mushroom molecular pharming relies on efficient transformation and gene expression.
Using strong promoters to express heterologous genes in appropriate hosts is a major strategy in biotechnological applications. The glyceraldehyde-3-phosphate dehydrogenase (GPD, EC 1.2.1.12) promoter is a strong constitutive promoter which can be induced by any carbon source and has been widely used in the expression of heterologous proteins in Saccharomyces cerevisiae, Pichia pastoris and other yeasts. GPD is one of the key enzymes in the glycolytic and gluconeogenesis pathway and comprises up to 5% of the soluble cellular protein content in S. cerevisiae and other higher eukaryotes. Furthermore, gpd mRNA accounts for 2-5% of the poly (A)+ RNA present in yeasts. These observations suggest that the gpd gene is regulated by a highly active promoter. In fact, vectors carrying the homologous gpd promoter region have been reported to be efficient in directing expression of heterologous genes in yeasts (Bitter and Egan, 1988, Doring et al., 1998, Eriksson et al., 1995, Vassileva et al., 2001) and filamentous fungi (Juge et al., 1998, Punt et al., 1987). However, known expression vectors containing genetic regulatory elements for expression in filamentous fungi of the ascomycetes class cannot be efficiently expressed in filamentous fungi of the basidiomycetes class.
The gpd genes have also been cloned from basidiomycetous fungi, including Schizophyllum commune, Phanerochaete chrysosporium, Agaricus bisporus (Harmsen et al., 1992), and Lentinula edodes. Among these mushrooms, genetic transformation using homologous gpd promoter was reported successful only in A. bisporus, Flammulina velutipes and L. edodes (Hirano et al., 2000, Kuo et al., 2004, van de Rhee et al., 1996). Although heterologous promoters have been used for the expression of drug-resistant marker genes, the genetic transformation is not sufficient to express heterologous genes. To sufficiently and effectively express a heterologous gene, it is important for a host cell to recognize the promoter sequence by its transcriptional machinery. Chun-Yi Kuo et al. demonstrated that a heterologous gene, hygromycin B phosphotransferase gene (hpt), can be expressed in F. velutipes (Kuo et al., 2004). However, it was found that the gpd genes in some basidiomycetous fungi, though highly similar, are significantly different in their promoter regions. U.S. Pat. No. 7,777,021 provides an isolated glyceraldehyde-3-phosphate dehydrogenase promoter in Pleurotus and a construct comprising the promoter operably linked to a heterologous transcribable polynucleotide molecule.
However, there is a need to increase gene expression of glyceraldehyde-3-phosphate dehydrogenase promoter in basidiomycetous fungi.